Mechanistic in vitro studies indicate that the clinical drug–drug interactions between protease inhibitors and rosuvastatin are driven by inhibition of intestinal BCRP and hepatic OATP1B1 with minimal contribution from OATP1B3, NTCP and OAT3

Abstract Previous use of a mechanistic static model to accurately quantify the increased rosuvastatin exposure due to drug–drug interaction (DDI) with coadministered atazanavir underpredicted the magnitude of area under the plasma concentration–time curve ratio (AUCR) based on inhibition of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1. To reconcile the disconnect between predicted and clinical AUCR, atazanavir and other protease inhibitors (darunavir, lopinavir and ritonavir) were evaluated as inhibitors of BCRP, OATP1B1, OATP1B3, sodium taurocholate cotransporting polypeptide (NTCP) and organic anion transporter (OAT) 3. None of the drugs inhibited OAT3, nor did darunavir and ritonavir inhibit OATP1B3 or NTCP. All drugs inhibited BCRP‐mediated estrone 3‐sulfate transport or OATP1B1‐mediated estradiol 17β‐D‐glucuronide transport with the same rank order of inhibitory potency (lopinavir>ritonavir>atazanavir>>darunavir) and mean IC50 values ranging from 15.5 ± 2.80 μM to 143 ± 14.7 μM or 0.220 ± 0.0655 μM to 9.53 ± 2.50 μM, respectively. Atazanavir and lopinavir also inhibited OATP1B3‐ or NTCP‐mediated transport with a mean IC50 of 1.86 ± 0.500 μM or 65.6 ± 10.7 μM and 5.04 ± 0.0950 μM or 20.3 ± 2.13 μM, respectively. Following integration of a combined hepatic transport component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above for atazanavir, the newly predicted rosuvastatin AUCR reconciled with the clinically observed AUCR confirming additional minor involvement of OATP1B3 and NTCP inhibition in its DDI. The predictions for the other protease inhibitors confirmed inhibition of intestinal BCRP and hepatic OATP1B1 as the principal pathways involved in their clinical DDI with rosuvastatin.

nect between predicted and clinical AUCR, atazanavir and other protease inhibitors (darunavir, lopinavir and ritonavir) were evaluated as inhibitors of BCRP, OATP1B1, OATP1B3, sodium taurocholate cotransporting polypeptide (NTCP) and organic anion transporter (OAT) 3. None of the drugs inhibited OAT3, nor did darunavir and ritonavir inhibit OATP1B3 or NTCP. All drugs inhibited BCRP-mediated estrone 3-sulfate transport or OATP1B1-mediated estradiol 17β-D-glucuronide transport with the same rank order of inhibitory potency (lopinavir>ritonavir>atazanavir>>darunavir) and mean IC 50 values ranging from 15.5 ± 2.80 μM to 143 ± 14.7 μM or 0.220 ± 0.0655 μM to 9.53 ± 2.50 μM, respectively. Atazanavir and lopinavir also inhibited OATP1B3-or NTCP-mediated transport with a mean IC 50 of 1.86 ± 0.500 μM or 65.6 ± 10.7 μM and 5.04 ± 0.0950 μM or 20.3 ± 2.13 μM, respectively. Following integration of a combined hepatic transport component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above for atazanavir, the newly predicted rosuvastatin AUCR reconciled with the clinically observed AUCR confirming additional minor involvement of OATP1B3 and NTCP inhibition in its DDI. The predictions for the other protease inhibitors confirmed inhibition of intestinal BCRP

| INTRODUC TI ON
Statins are widely prescribed for the effective treatment of hypercholesterolemia and as a drug class are generally well tolerated in humans. 1 However, in up to 29% of patients, adverse effects associated with myopathy have been reported with their use, ranging from mild muscle pain to (in extreme cases) fatal rhabdomyolysis. 2 Because of this it is imperative that careful consideration be given to factors that may have an impact on the disposition and therefore exposure of statins. Consequently, drug-drug interactions (DDIs) with statins are of clinical concern as elevated plasma concentrations of statins, due to inhibition of critical disposition pathways by a co-medication, are associated with increased muscle exposure and therefore risk of myopathy. 3 Statins are increasingly being prescribed in human immunodeficiency virus (HIV)-infected patients since dyslipidemias are common comorbidities of HIV infection, 4 as identified in 24% of patients. 5 As a result of their subsequent widespread use in HIV patients, 6 it should not come as a surprise that clinically significant DDIs resulting in myotoxicities have been observed between antiretroviral protease inhibitors and statin drugs. 7 A particular concern is the potential for DDI between protease inhibitors and the hydrophilic statin rosuvastatin, which has been shown to be more effective in managing dyslipidemia in HIV patients than other statins and has one of the largest prescription frequencies amongst the statins. 6,8 In fact, clinically significant DDIs resulting in up to 3-fold increases in rosuvastatin exposure (defined as area under the plasma concentration-time curve, AUC) have been observed between rosuvastatin and the perpetrators atazanavir, darunavir or lopinavir/ritonavir (Crestor® drug label) [9][10][11][12] . The critical disposition pathways of rosuvastatin and their contributions to overall clearance (fraction excreted [ƒ e ] values) have been determined previously. 1 These critical pathways include intestinal breast cancer resistance protein (BCRP) efflux as the barrier to absorption (fraction absorbed = 0.5, therefore BCRP ƒ e = 0.5), hepatic organic anion transporting polypeptide 1B1 (OATP1B1, ƒ e = 0.38) uptake for hepatic elimination, and active renal secretion of rosuvastatin mediated by organic anion transporter 3 (OAT3, ƒ e = 0.25) uptake. Other minor pathways that contribute toward the overall hepatic elimination of rosuvastatin (ƒ e = 0.7) include OATP1B3 (ƒ e = 0.11) and sodium taurocholate cotransporting polypeptide (NTCP, ƒ e = 0.21). 13 Although the mechanisms underlying rosuvastatin-protease inhibitor DDIs are not yet fully characterized, studies by Annaert et al., 14 Weiss et al. 15 and Elsby et al. 13 established that many protease inhibitors are in vitro inhibitors of the key disposition transporters BCRP and OATP1B1. Moreover, Elsby et al. 13 used their published mechanistic static model to predict accurately the AUC ratio (AUCR) of rosuvastatin due to inhibition of BCRP and OATP1B1 pathways by darunavir and lopinavir, establishing that these specific DDIs are primarily driven by perturbation of BCRP function, and to a lesser extent by OATP1B1. However, for atazanavir, inhibition of BCRP and OATP1B1 alone could not fully reconcile its clinically observed DDI with rosuvastatin (predicted AUCR = 2.13 versus clinical AUCR = 3.1). Only when the overall hepatic uptake pathway (ƒ e = 0.7) was considered using a literature OATP1B1 K i as presumed surrogate for inhibition of "overall active hepatic uptake", did the mechanistic approach predict (AUCR = 3.25) the clinical observation. 16 The assumptions made for that prediction were that atazana-

| Assessment of BCRP inhibition (IC 50 determination)
The methodology used was adapted from the previously validated Caco-2 unidirectional (basolateral to apical; B-A) BCRP inhibition assay. 13 Briefly, Caco-2 cells between passage numbers 40-60 were seeded onto Millicell-96 multiwell cell culture insert plates at 1 × 10 5 cells/cm 2 and cultured at 37°C in an atmosphere of 5 % CO 2 with a relative humidity of 95%. The cells were cultured in medium (consisting of DMEM supplemented with 10% (w/v) fetal bovine serum, 2 mM L-glutamine, 1% (v/v) nonessential amino acids and 50 U mL −1 penicillin and 50 μg mL −1 streptomycin) which was changed every two or three days. quently converted to concentration (pmol/mL) and used to calculate apparent permeability (P app ), as described previously. 18 The passive permeability of estrone 3-sulfate observed when BCRP is completely inhibited (derived from incubations containing the highest concentration (100 μM) of the positive control inhibitor novobiocin) was subtracted from the determined B-A P app value in the absence or presence of test inhibitor, to give a corrected BCRP-mediated B-A P app , which was subsequently converted to percentage (vehicle) control transport activity. For each test inhibitor, determined percentage control transport activity was plotted against nominal inhibitor concentration and fitted using SigmaPlot 12.5 (Systat Software Inc., Chicago, IL; four parameter logistic equation) to determine the concentration that produces half-maximal inhibition of probe substrate transport (IC 50 ; equivalent to K i as probe substrate concentration in the assay is at least 7-times lower than its K m , and assuming competitive inhibition). The P app of co-incubated lucifer yellow across the cell monolayer was determined and cell monolayer integrity was deemed acceptable if P app < 1.0 × 10 −6 cm/s.

| Assessment of SLC transporter inhibition (single concentration screen)
OATP1B3, NTCP, OAT3 and vector control cell lines were seeded in cell culture medium (consisting of DMEM supplemented with 10% (w/v) fetal bovine serum and 1% (v/v) nonessential amino acids) into 24-well poly-D-lysine coated plates at 3-4 × 10 5 cells per well to achieve a pre-assay confluence of typically 80%-95%. Cells were cultured at 37°C, 8% CO 2 for 24 h and media was replaced 3-4 h post-seeding with either fresh media for OAT3 cells (and corresponding control cells) or fresh media containing 2 mM sodium butyrate for all other cell lines. Prior to assay, cells were washed twice with pre-warmed uptake buffer (HBSS containing 10 mM HEPES, pH 7.4) then pre-incubated with test inhibitor drug (at the single concentration stated below) at 37°C in warm uptake buffer for 15 min. After the pre-incubation step, uptake buffer was removed and the appropriate corresponding incubation solutions were added to the wells. For data analysis, the determined total uptake of radiolabeled probe substrate into cells (pmol) was normalized to the protein (mg) content of each well to calculate the uptake activity (pmol/mg).
Uptake activity of probe substrate into vector control cells was subtracted from that determined into transporter-expressing cells to calculate the corrected transporter-mediated uptake. Corrected uptake activity (pmol/mg) was subsequently converted to percentage (vehicle) control transport activity.

| Assessment of SLC transporter inhibition (IC 50 determination)
OATP1B1, OATP1B3, NTCP and vector control cell lines were cultured and subsequent transporter assays conducted as described above using the same transporter-specific probe substrates and incubation times. For OATP1B1, the probe substrate was For each drug, determined percentage (vehicle) control transport activity was plotted against nominal inhibitor concentration and fitted using SigmaPlot 12.5 (Systat Software Inc., Chicago, IL; four parameter logistic equation) to determine the concentration that produces half-maximal inhibition of probe substrate transport (IC 50 ; equivalent to K i assuming competitive inhibition as probe substrate concentration in the assay is at least 10-times lower than its K m ).  Transporters 1+2+3) transporters, 19 or [I] = unbound maximum plasma concentration at steady state (C max u = f u × C max ) for renal transporters. 19 f u = unbound fraction in plasma, C max = maximum total plasma concentration of inhibitor at steady state, F a F g = fraction of the dose absorbed after oral administration, k a = absorption rate constant (min −1 ), Q h = hepatic blood flow (1617 mL/min), R B is the blood-toplasma concentration ratio (default = 1.0) and Q ent = enterocyte blood flow (300 mL/min).

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy.

| Predicted versus observed rosuvastatin AUC changes with protease inhibitors based upon in vitro transporter inhibitory data
Using the mean drug inhibitory parameters determined above for atazanavir, darunavir, lopinavir and ritonavir versus BCRP, OATP1B1, OATP1B3 or NTCP and their pharmacokinetic parameters provided in Table 5, calculations were performed with mechanistic static equations (1 and 2) in order to predict the theoretical fold-increase in rosuvastatin AUC that would occur following co-administration.

F I G U R E 1 Representative plot of mean concentration-dependent inhibition of BCRP-mediated transport of [ 3 H]-estrone 3-sulfate (1 μM)
by atazanavir, darunavir, lopinavir or ritonavir. Data are expressed as mean (± standard deviation) from 12 wells per incubation condition (triplicate wells on four separate occasions) for atazanavir and ritonavir, or 9 wells per incubation condition (triplicate wells on three separate occasions) for darunavir and lopinavir.
Calculated AUC ratios (AUCR) due to inhibition of each transporter alone, and in combination, are shown in Note: K i = absolute inhibition constant versus the transporter (assuming competitive inhibition, this equates to IC 50 in the assays as probe substrate concentration utilized is <<<< K m ). a 95% confidence interval was calculated by adding or subtracting the product of the t-distribution value (4.303) and the value of the standard deviation divided by the square root of the sample size from the determined mean value. Note: K i = absolute inhibition constant versus the transporter (assuming competitive inhibition, this equates to IC 50 in the assays as probe substrate concentration utilized is <<<< K m ). a 95% confidence interval was calculated by adding or subtracting the product of the t-distribution value (4.303) and the value of the standard deviation divided by the square root of the sample size from the determined mean value.  For assessment of inhibition of BCRP function, the present study has utilized the regulatory industry gold standard methodology involving the polarized Caco-2 cell monolayer test system, which has previously been extensively validated to correctly identify inhibitors of BCRP using rosuvastatin as probe substrate. 13 In this study, the prototypical BCRP substrate estrone 3-sulfate was used as a good surrogate in vitro probe substrate for rosuvastatin since established BCRP inhibitors gave similar K i values to those obtained with rosuvastatin. 16 The reason why estrone 3-sulfate is a good surrogate is due to the similar hydrophilic properties it shares with rosuvastatin (existing predominantly in the charged anionic form at physiological pH) resulting in its subsequent mechanistic translatability to rosuvastatin F I G U R E 4 Representative plot of mean concentration-dependent inhibition of OATP1B3-mediated transport of [ 3 H]estradiol 17β-D-glucuronide (0.02 μM) by atazanavir or lopinavir. Data are expressed as mean (± standard deviation) from 9 wells per incubation condition (triplicate wells on three separate occasions).

TA B L E 4 Determined in vitro K i (IC 50 ) values for inhibition of BCRP and OATP1B1 by ritonavir
vectorial (basolateral to apical) transport processes in Caco-2 cells.
Due to their negligible intrinsic passive membrane permeability, both estrone 3-sulfate and rosuvastatin require an active basolateral uptake process in order to enter cells to interact with apically expressed BCRP (for which they are selective substrates). In Caco-2 cells this uptake is mediated by the passive facilitative organic solute transporter alpha/beta (OSTα/β 28,29 ). Indeed, it is because of this shared mechanistic property that employing an alternative polarized cell system overexpressing BCRP (i.e., MDCK-BCRP) is actually detrimental to the use of clinically relevant rosuvastatin, or its surrogate estrone F I G U R E 5 Representative plot of mean concentration-dependent inhibition of NTCP-mediated transport of [ 3 H]taurocholic acid (1 μM) by atazanavir or lopinavir. Data are expressed as mean (± standard deviation) from 9 wells per incubation condition (triplicate wells on three separate occasions).
3-sulfate, as probe substrate for BCRP function due to the fact that MDCK-BCRP cells are deficient in the basolateral uptake component, thereby hindering their entry into cells. In contrast to Caco-2, this deficiency in MDCK-BCRP precludes its use as a test system for the correct identification of highly polar substrates or inhibitors of BCRP. 29 This also explains why MDCK-BCRP assays require the use of more permeable/lipophilic BCRP probe substrates (e.g., cladribine, prazosin) in order to observe good transport function which, unfortunately, are not relatable to hydrophilic rosuvastatin as the major victim of clinically significant BCRP-mediated DDIs. 16 Regarding the Caco-2 test system, the similarity and ranking of K i values for the known BCRP inhibitors novobiocin, Ko143, cyclosporin A, pantoprazole, sulfasalazine, atorvastatin, diclofenac, fluvastatin and nifedipine obtained in the previous validation with corresponding literature values derived from inside-out membrane vesicles expressing BCRP, 13 gives confidence that the mechanism of inhibition observed in the Caco-2 test system reflects perturbation of BCRP efflux rather than of basolateral uptake and, as such, the resulting determined K i is the correct one for assessing DDI risk at BCRP.
The present study confirmed that all the protease inhibitor drugs  (Tables 1-4), 13,15,30 confirming the suitability of estrone 3-sulfate as a good surrogate in vitro probe substrate for rosuvastatin. 16 Furthermore, the determined mean OATP1B1 IC 50 (K i ) values were also within 1.2-to 2-fold of previous studies that utilized either the same recommended surrogate OATP1B1 in vitro probe substrate estradiol 17β-D-glucuronide 26,27 or the clinically relevant probe atorvastatin, for atazanavir, darunavir or lopinavir (Tables 1-3). 13,31 For investigations involving OAT3, NTCP and OATP1B3, initial inhibition experiments were performed using a single inhibitor con-  In the case of darunavir, since it is not an inhibitor of OATP1B3 or NTCP, the mechanism for perpetrating its DDI is principally inhibition of BCRP and, to a lesser extent, inhibition of OATP1B1. The AUCR predictions in this study are marginally lower than that of Elsby et al. 13 and likely reflect the 2-fold higher BCRP K i determined here (reduc-

ACK N O WLE D G E M ENTS
We would like to thank Gabrielle Vanden, John Mombili and Millie Turner for their assistance in data checking the processed experimental results.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors are all employees, or a former employee (HC), of Cyprotex Discovery Ltd (an Evotec Company) which is a contract research organization.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data that support the findings of this study are available from the corresponding author upon reasonable request.